Authors:Andrew D. Tustian; Linus Laurin, Henrik Ihre, Travis Tran, Robert Stairs, Hanne BakAbstract: There is strong interest in the production of bispecific monoclonal antibodies that can simultaneously bind two distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Regeneron's bispecific technology, based upon a standard IgG, consists of a heterodimer of two different heavy chains, and a common light chain. Co-expression of two heavy chains leads to the formation of two parental IgG impurities, the removal of which is facilitated by a dipeptide substitution in the Fc portion of one of the heavy chains that ablates Fc Protein A binding. Therefore the affinity capture (Protein A) step of the purification process must perform both bulk capture and high resolution of these mAb impurities, a task current commercially available resins are not designed for. Resolution can be further impaired by the ability of Protein A to bind some antibodies in the variable region of the heavy chain (VH). This paper details development of a novel Protein A resin. This resin combines an alkali stable ligand with a base matrix exhibiting excellent mass transfer properties to allow high capacity single step capture and resolution of bispecific antibodies with high yields. The developed resin, named MabSelect SuRe™ pcc, is implemented in GMP production processes for several bispecific antibodies. This article is protected by copyright. All rights reserved.PubDate: 2018-02-21T03:45:48.28025-05:0DOI: 10.1002/btpr.2622

Authors:Amish Asthana; Charles McRae White, Megan Douglass, William S. KisaalitaAbstract: The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug-induced liver injury (DILI), and has led to a demand for robust, human cell-based, in vitro assays for drug discovery. Microporous polymer-based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high-throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi-like structures. Poly-L Lysine (PLL) and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 μm; B: 175 ± 6 μm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost-effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer-based scaffolds suitable for drug discovery. This article is protected by copyright. All rights reserved.PubDate: 2018-02-21T03:42:11.277419-05:DOI: 10.1002/btpr.2627

Authors:Ahasanul Karim; Abu Yousuf, M Amirul Islam, Yasir H Naif, Che Ku Mohammad Faizal, Md. Zahangir Alam, Domenico PirozziAbstract: The aim of the study was to investigate the feasibility of using irreversible electroporation (EP) as a microbial cell disruption technique to extract intracellular lipid within short time and in an eco—friendly manner. An EP circuit was designed and fabricated to obtain 4 kV with frequency of 100 Hz of square waves. The yeast cells of Lipomyces starkeyi (L. starkeyi) were treated by EP for 2-10 min where the distance between electrodes was maintained at 2, 4 and 6 cm. Colony forming units (CFU) were counted to observe the cell viability under the high voltage electric field. The forces of the pulsing electric field caused significant damage to the cell wall of L. starkeyi and the disruption of microbial cells was visualized by field emission scanning electron microscopic (FESEM) image. After breaking the cell wall, lipid was extracted and measured to assess the efficiency of EP over other techniques. The extent of cell inactivation was up to 95% when the electrodes were placed at the distance of 2 cm, which provided high treatment intensity (36.7 kWh/m3). At this condition, maximum lipid (63 mg/g) was extracted when the biomass was treated for 10 min. During the comparison, EP could extract 31.88% lipid while the amount was 11.89% for ultrasonic and 16.8% for Fenton's reagent. The results recommend that the EP is a promising technique for lowering the time and solvent usage for lipid extraction from microbial biomass. This article is protected by copyright. All rights reserved.PubDate: 2018-02-21T03:41:55.643522-05:DOI: 10.1002/btpr.2625

Authors:Seo-Young Park; Thomas M. Reimonn, Cyrus Agarabi, Kurt Brorson, Seongkyu YoonAbstract: Amino acids and glucose consumption, cell growth and monoclonal antibody (mAb) production in mammalian cell culture are key considerations during upstream process and particularly media optimization. Understanding the interrelations and the relevant cellular physiology will provide insight for setting strategy of robust and effective mAb production. The aim of this study was to further our understanding of nutrient consumption metabolism, since this could have significant impact on enhancing mAb titer, cell proliferation, designing feeding strategies, and development of feed media. The nutrient consumption pattern, mAb concentration, and cell growth were analyzed in three sets of cell cultures with media supplementation of glucose, methionine, threonine, tryptophan, and tyrosine. The amino acids metabolism and its impact on cell growth and mAb production during the batch and fed-batch culture were closely analyzed. It was shown that the phenylalanine, tyrosine and tryptophan biosynthesis pathways were significantly altered under different culture conditions with different media. These changes were more apparent in the fed-batch process in which higher mAb titer was observed due to the metabolic changes than mAb titer in the batch process. The pathway analysis approach was well utilized for evaluating the impact on the relevant pathways involved under different cell culture conditions to improve cell growth and mAb titer. This article is protected by copyright. All rights reserved.PubDate: 2018-02-21T03:41:44.855678-05:DOI: 10.1002/btpr.2623

Authors:Yi Feng Lee; Matthias Jöhnck, Christian FrechAbstract: The efficiencies of mono gradient elution and dual salt-pH gradient elution for separation of six mAb charge and size variants on a preparative-scale ion exchange chromatographic resin are compared in this study. Results showed that opposite dual salt-pH gradient elution with increasing pH gradient and simultaneously decreasing salt gradient is best suited for the separation of these mAb charge and size variants on Eshmuno® CPX. Besides giving high binding capacity, this type of opposite dual salt-pH gradient also provides better resolved mAb variant peaks and lower conductivity in the elution pools compared to single pH or salt gradients. To have a mechanistic understanding of the differences in mAb variants retention behaviors of mono pH gradient, parallel dual salt-pH gradient, and opposite dual salt-pH gradient, a linear gradient elution model was used. After determining the model parameters using the linear gradient elution model, 2-D plots were used to show the pH and salt dependencies of the reciprocals of distribution coefficient, equilibrium constant, and effective ionic capacity of the mAb variants in these gradient elution systems. Comparison of the 2-D plots indicated that the advantage of opposite dual salt-pH gradient system with increasing pH gradient and simultaneously decreasing salt gradient is the non-continuous increased acceleration of protein migration. Furthermore, the fitted model parameters can be used for the prediction and optimization of mAb variants separation in dual salt-pH gradient and step elution. This article is protected by copyright. All rights reserved.PubDate: 2018-02-21T03:41:33.715414-05:DOI: 10.1002/btpr.2626

Authors:B.S. Priyanka; Navin K. RastogiAbstract: The present work deals with the extraction of lipase and amylase from enzyme mixture by employing liquid emulsion membranes (LEM). The electrostatic interaction between enzymes and reverse micellar surfactant polar head group plays an important role for selective extraction of two different enzymes having different isoelectric points. The optimised conditions for lipase extraction (pH 7.0) resulted in the purification fold and activity recovery of 5.43 fold and 89.53%, respectively, whereas, in case of amylase (pH 9.0) the purification fold and activity recovery were 6.58 and 94.32%, respectively. The results were compared with the control sample (containing individual enzymes) and mixture of enzymes lipase and amylase and it was shown that for optimum conditions the activity recovery and purification fold was higher for the individual enzymes as compared to their mixture. Downstream processing involving LEM was shown to be a feasible method for selective extraction of enzymes. This article is protected by copyright. All rights reserved.PubDate: 2018-02-21T03:41:15.850412-05:DOI: 10.1002/btpr.2624

Authors:Sen Xu; Rubin Jiang, Roland Mueller, Nadja Hoesli, Thomas Kretz, John Bowers, Hao ChenAbstract: Lactate metabolism variations are frequently encountered in mammalian cell culture processes, especially during process scale-up. In this study, we took a multipronged approach to investigate the impact of pH, pCO2, osmolality, base addition, and mixing conditions on the observed lactate variations in a Chinese Hamster Ovary (CHO) fed-batch process at 2,000 L scale. Two cultivating methods, CO2-controlled and pH-controlled, were used to decouple the individual and synergistic effects from those factors. The individual effects from pH, pCO2, and osmolality on lactate consumption/reproduction in the stationary phase were insignificant in the ranges studied though the initial lactate production rates varied. In contrast, lactate metabolism was found to be impacted by an interaction between mixing conditions and CO2 accumulation. High CO2 accumulation and poor mixing led to lactate reproduction, whereas either low CO2 or improved mixing were sufficient to result in lactate consumption. Base addition was not required for pH control in the low CO2 conditions, and therefore lactate reproduction was correlated with base addition under poor mixing conditions. Under good mixing conditions, CO2-triggered base addition did not significantly impact lactate reproduction. It is reasonable to postulate that increased mixing times further promoted lactate production during base addition. As lactate reproduction results in more base addition to maintain pH, a cycle could be formed between lactate production and base addition. As a remediation, we showed that such lactate reproduction could be eliminated by improving CO2 removal at 2,000 L scale. This article is protected by copyright. All rights reserved.PubDate: 2018-02-21T03:40:22.685537-05:DOI: 10.1002/btpr.2620

Authors:Andrea Svoradová; Lenka Kuželová, Jaromír Vašíček, Lucia Olexíková, Peter ChrenekAbstract: The goal of this study was to evaluate effect of slow freezing and vitrification methods on the viability of chicken blastodermal cells (BCs). Proper aliquot of isolated BCs were diluted in the freezing medium composed of 10% DMSO and frozen in the freezing vessel BICELL to reach desired temperature up to −80°C. Then samples were immersed in liquid nitrogen. Other cell aliquot was vitrified in solution containing 10% DMSO and samples were immediately immersed in the liquid nitrogen. The viability of fresh and frozen/thawed BCs was evaluated using Trypan blue method and flow cytometry. Flow cytometry analysis was provided by DRAQ5 dye in combination with Live-Dead kit. Overall, this technique provides both quantitative and qualitative information about BCs. Results obtained from Trypan blue method showed significant differences (P PubDate: 2018-02-12T01:31:22.342823-05:DOI: 10.1002/btpr.2615

Authors:Kseniya A. Sheshukova; Lisa R. WilkenAbstract: Transgenic plant systems have successfully been used to express recombinant proteins, including rice seed-expressed recombinant human serum albumin (rHSA), without the risk of contamination of human pathogens. Developing an efficient extraction process is critical as the step determines recombinant protein concentration and purity, quantity of impurities, and process volume. This paper evaluates the effect of pH and time on the extraction and stability of rHSA. The amount of rHSA in clarified extract after 60 min of solubilization increased with pH from 0.9 mg/g (pH 3.5) to 9.6 mg/g (pH 6.0), but not over time as 10 min was sufficient for solubilization. Total soluble protein (TSP) in extracts also increased with pH from 3.9 mg/g (pH 3.5) to 19.7 mg/g (pH 6.0) in clarified extract. Extraction conditions that maximized rHSA purity were not optimal for rHSA stability and yield. Extraction at pH 3.5 resulted in high purity (78%), however, rHSA degraded over time. Similar purities (78%) were observed in pH 4.0 extracts yet rHSA remained stable. rHSA degradation was not observed in pH 4.5 and 6.0 extracts but higher native protein concentrations decreased purity. Strategies such as pH and temperature adjustment were effective for reducing rHSA degradation in pH 3.5 rice extracts. Low temperature pH 3.5 extraction retained high purity (97%) and rHSA stability. While seed-expressed recombinant proteins are known to be stable for up to 3 years, the degradation of rHSA was notably extensive (56% within 60 min) when extracted at low pH. This article is protected by copyright. All rights reserved.PubDate: 2018-01-08T03:33:29.21138-05:0DOI: 10.1002/btpr.2609

Authors:Agnieszka Kołodziejczak-Radzimska; Jakub Zdarta, Teofil JesionowskiAbstract: Acylase I from Aspergillus melleus was immobilized on supports consisting of unmodified and modified silica. Modification was performed using 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA). The effectiveness of immobilization was investigated using the standard Bradford method in addition to a number of physicochemical techniques, including spectroscopic methods (FTIR, 29Si and 13C CP MAS NMR), porous structure and elemental analysis, and zeta potential measurement. A determination of catalytic activity was made based on the deacetylation reaction of N-acetyl-l-methionine. Furthermore, the effect of pH and temperature on the catalytic activity of the free and immobilized enzyme, as well as the reusability of the silica-bound aminoacylase, were determined. The immobilized systems demonstrated a high degree of catalytic activity. The best catalytic parameters were those of aminoacylase immobilized on silica modified with APTES (apparent activity 3937 U/g, relative activity 61.6%). This article is protected by copyright. All rights reserved.PubDate: 2018-01-08T03:33:19.2538-05:00DOI: 10.1002/btpr.2610

Authors:Jian Dong; Kun-Qiang Hong, Ai-Li Hao, Cui-Ying Zhang, Xiao-Meng Fu, Peng-Fei Wang, Dong-Guang XiaoAbstract: As content and proportion of ethyl acetate is critical to the flavor and quality of beverages, the concise regulation of the ethyl acetate metabolism is a major issue in beverage fermentations. In the present study, for ethyl acetate yield regulation, we finely modulated the expression of ATF1 through precise and seamless insertion of serially truncated PGK1 promoter from the 3'end by 100bp steps in the Chinese liquor yeast, CLy12a. The three engineered promoters carrying 100-, 200- and 300-bp truncations exhibited reduced promoter strength but unaffected growth. These three promoters were integrated into the CLy12a strain, generating strains CLy12a-P-100, CLy12a-P-200 and CLy12a-P-300, respectively. The transcription levels of CLy12a-P-100, CLy12a-P-200 and CLy12a-P-300 were 20%, 17% and 10% of that of CLy12a-P, respectively. The AATase (Alcohol acetyl transferases, encoded by the ATF1 gene) activity of three engineered strains were 36%, 56% and 62% of that of CLy12a-P. In the liquid fermentation of corn hydrolysate at 30°C, the concentration of ethyl acetate in CLy12a-P-100, CLy12a-P-200 and CLy12a-P-300 were reduced by 28%, 30% and 42%, respectively, compared to CLy12a-P. These results verifying that the ethyl acetate yield could be gradually enhanced by finely modulating the expression of ATF1. The engineered strain CLy12a-P-200 produced the ethyl acetate concentration with the best sensorial quality compared to the other engineered yeast strains. The method proposed in this work supplies a practical proposal for breeding Chinese liquor yeast strains with finely modulated ethyl acetate yield. This article is protected by copyright. All rights reserved.PubDate: 2018-01-05T03:35:54.512383-05:DOI: 10.1002/btpr.2605

Authors:Eric Karengera; Anna Robotham, John Kelly, Yves Durocher, Gregory De Crescenzo, Olivier HenryAbstract: Lactate and ammonia accumulation is a major factor limiting the performance of fed-batch strategies for mammalian cell culture processes. In addition to the detrimental effects of these by-products on production yield, ammonia also contributes to recombinant glycoprotein quality deterioration. In this study, we tackled the accumulation of these two inhibiting metabolic wastes by culturing in glutamine-free fed-batch cultures an engineered HEK293 cell line displaying an improved central carbon metabolism. Batch cultures highlighted the ability of PYC2-overexpressing HEK293 cells to grow and sustain a relatively high viability in absence of glutamine without prior adaptation to the culture medium. In fed-batch cultures designed to maintain glucose at high concentration by daily feeding a glutamine-free concentrated nutrient feed, the maximum lactate and ammonia concentrations did not exceed 5 mM and 1 mM, respectively. In flask, this resulted in more than a 2.5-fold increase in IFNα2b titer in comparison to the control glutamine-supplied fed-batch. In bioreactor, this strategy led to similar reductions in lactate and ammonia accumulation and an increase in IFNα2b production. Of utmost importance, this strategy did not affect IFNα2b quality with respect to sialylation and glycoform distribution as confirmed by surface plasmon resonance biosensing and LC-MS, respectively. Our strategy thus offers an attractive and simple approach for the development of efficient cell culture processes for the mass production of high quality therapeutic glycoproteins. This article is protected by copyright. All rights reserved.PubDate: 2018-01-05T03:35:52.157494-05:DOI: 10.1002/btpr.2607

Authors:Meng-Yao Li; Bruno Ebel, Cédric Paris, Fabien Chauchard, Emmanuel Guedon, Annie MarcAbstract: The glycosylation of therapeutic monoclonal antibodies (mAbs), a known critical quality attribute, is often greatly modified during the production process by animal cells. It is essential for biopharmaceutical industries to monitor and control this glycosylation. However, current glycosylation characterization techniques involve time- and labor-intensive analyses, often carried out at the end of the culture when the product is already synthesized. This study proposes a novel methodology for real-time monitoring of antibody glycosylation site occupancy using Raman spectroscopy. It was first observed in CHO cell batch culture that when low nutrient concentrations were reached, a decrease in mAb glycosylation was induced, which made it essential to rapidly detect this loss of product quality. By combining in situ Raman spectroscopy with chemometric tools, efficient prediction models were then developed for both glycosylated and non-glycosylated mAbs. By comparing Variable Importance in Projection profiles of the prediction models, it was confirmed that Raman spectroscopy is a powerful method to distinguish extremely similar molecules, despite the high complexity of the culture medium. Finally, the Raman prediction models were used to monitor batch and feed-harvest cultures in situ. For the first time, it was demonstrated that the concentrations of glycosylated and non-glycosylated mAbs could be successfully and simultaneously estimated in real-time with high accuracy, including their sudden variations due to medium exchanges. Raman spectroscopy can thus be considered as a promising PAT tool for feedback process control dedicated to on-line optimization of mAb quality. This article is protected by copyright. All rights reserved.PubDate: 2018-01-05T03:35:43.548707-05:DOI: 10.1002/btpr.2604

Authors:Mili Pathak; Katherine Lintern, Daniel G. Bracewell, Anurag S. RathoreAbstract: Adsorbent lifetime during protein A chromatography is not readily predicted or understood, representing a key challenge to be addressed for biopharmaceutical manufacturers. This paper focuses on the impact of feed composition on the performance of a typical agarose based protein A resin across a lifetime of 50 cycles. Cycling studies were performed using three different feed materials with varying levels of feed components including proteases, histones, DNA, and non-histone proteins. Changes in the process and quality attributes were measured. The DBCs were not seen to vary between conditions although there was a reduction in particle porosity in all cases. Fluorescence spectroscopy and LC-MS/MS were used to identify the contribution and extent of fouling to the observed capacity loss. Residual protein A ligand density and deposition of foulants (HCP, residual mAb and DNA) varied between the three feed materials. Resins cycled in feed materials containing high concentrations of HCP and histones were seen to have greater extents of capacity loss. The mode of performance loss, capacity loss or impact on product quality was seen to vary depending upon the feed material. The results indicate that feed material composition may be correlated to the rate and mode of resin ageing as a basis for improved process understanding. This article is protected by copyright. All rights reserved.PubDate: 2018-01-05T03:35:36.320702-05:DOI: 10.1002/btpr.2608

Authors:Likuan Zhu; Xueting Zhang, Kai Cheng, Zhonghua Lv, Lei Zhang, Qingyong Meng, Shujie Yuan, Boyan Song, Zhenlong WangAbstract: We conducted a three-dimensional computational fluid dynamics (CFD) simulation to calculate the flow field in the Inverted Frusto-conical Shaking Bioreactor with 5 L working volume (IFSB-5L). The CFD models were established for the IFSB-5L at different operating conditions (different shaking speeds and filling volumes) and validated by comparison of the liquid height distribution in the agitated IFSB-5L. The “out of phase” operating conditions were characterized by analyzing the flow field in the IFSB-5L at different filling volumes and shaking speeds. The values of volumetric power consumption (P/VL) and volumetric mass transfer coefficient (kLa) were determined from simulated and experimental results respectively. Finally, the operating condition effect on P/VL and kLa was investigated. This article is protected by copyright. All rights reserved.PubDate: 2018-01-05T03:30:53.551954-05:DOI: 10.1002/btpr.2602

Authors:Jayashree Subramanian; Rigzen P. S. Aulakh, Parbir S. Grewal, Mark Sanford, Abigail F. J. Pynn, Inn H. YukAbstract: Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO-based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell-specific productivity (Qp) over time for recombinant CHO cells developed using the glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze-thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze-thaw did not result in any significant impact beyond the initial post-thaw passages. Production cultures sourced from cryopreserved cells and their non-cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Qp at increased cell age: 17 of the 24 cell lines displayed ≥ 20% Qp decline after ∼2-3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Qp decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze-thaw as a cause for Qp decline. This article is protected by copyright. All rights reserved.PubDate: 2018-01-05T03:30:30.411462-05:DOI: 10.1002/btpr.2599

Authors:Ana M. Almeida; Joana Tomás, Patrícia Pereira, João A. Queiroz, Fani Sousa, Ângela SousaAbstract: DNA vaccines have come to light in the last decades as an alternative method to prevent many infectious diseases, but they can also be used for the treatment of specific diseases, such as cervical cancer caused by Human Papillomavirus (HPV). This virus produces E6 and E7 oncoproteins, which alter the cell cycle regulation and can interfere with the DNA repairing system. These features can ultimately lead to the progression of cervical cancer, after cell infection by HPV. Thus, the development of a DNA vaccine targeting both proteins arises as an interesting option in the treatment of this pathology. Nonetheless, before evaluating its therapeutic potential, the purity levels of a biopharmaceutical must meet the regulatory agency specifications. Previously, our research group successfully purified the supercoiled isoform of the recombinant HPV-16 E6/E7 DNA vaccine with virtual 100% purity by affinity chromatography. The present work was designed to evaluate the effect that pDNA sample purity levels may exert in the expression of a target protein. Thus, in vitro studies were performed to assess the vaccine ability to produce the target proteins and to compare the expression efficiency between the pDNA sample obtained by affinity chromatography, which only presents the sc isoform and fulfils the regulatory agency recommendations, and the same DNA vaccine retrieved by a commercial purification kit, which contains different pDNA isoforms. Our achievements suggest that the E6/E7 DNA vaccine purified by affinity chromatography promotes higher E6 and E7 mRNA and protein expression levels than the DNA vaccine purified with the commercial kit. Overall, these results underline the importance that a purification strategy may present in the therapeutic outcome of recombinant DNA vaccines, envisaging their further application as biopharmaceuticals. This article is protected by copyright. All rights reserved.PubDate: 2018-01-05T03:30:27.229608-05:DOI: 10.1002/btpr.2603

Authors:Najmeh Alizadeh; Vajihe Akbari, Maryam Nurani, Azade TaheriAbstract: Cellulose nanofibers (Cel-NFs) gel can be considered as a useful drug carrier because of its biocompatibility, high specific surface area and high loading capacity of drugs. Injectable Cel-NFs gel could deliver doxorubicin (DOX) for localized chemotherapy of melanoma and suppress melanoma cells migration because of the physical barrier property of Cel-NFs. We prepared DOX surface modified Cel-NFs (DOX-Cel-NFs) gel by the electrostatic attachment of DOX molecules on the surface of Cel-NFs. The increase in the zeta potential of nanofibers and the changes in the FTIR spectra of DOX-Cel-NFs compared to Cel-NFs proved this attachment. DOX-Cel-NFs showed nano-fibrous structure with an average diameter of 22.32 ±10.66 nm after analyzing using FESEM. The suitable injectability of DOX-Cel-NFs gel verified its promising application for the localized chemotherapy. DOX-Cel-NFs gel exhibited a sustained drug release manner. The cytotoxicity results showed that DOX-Cel-NFs were more cytotoxic against melanoma cancer cells than the free DOX during 48 h incubation period. Moreover, DOX-Cel-NFs gel can suppress the melanoma cancer cells migration efficiently. Thus our results emphasize the potential of DOX-Cel-NFs gel as a chemotherapeutic agent for local delivery of DOX in order to treat melanoma and prevent its metastasis. This article is protected by copyright. All rights reserved.PubDate: 2018-01-05T03:30:25.0006-05:00DOI: 10.1002/btpr.2598

Authors:Alex Brinkmann; Sanaa Elouafiq, John Pieracci, Matthew WestobyAbstract: Decoupling upstream and downstream operations in biopharmaceutical production could enable more flexible manufacturing operations and could allow companies to leverage strategic or financial benefits that would be otherwise unattainable. A decoupling process was developed and scaled-up utilizing single pass tangential flow filtration for volume reduction followed by bulk freezing in single-use bags prior to purification. Single pass tangential flow filtration can be used to continuously concentrate harvested cell culture fluid, reducing the volume by 15-25X with a step yield of greater than 96%. These concentration factors were reproduced with a second product, indicating that the process could be amenable to platform processes. Experimental data indicates that the product tested was stable for at least one year at -40 or -70°C. The concentration of the harvested cell culture fluid, either with or without a subsequent period of frozen storage, had no impact on the product quality attributes that were tested. This article is protected by copyright. All rights reserved.PubDate: 2018-01-05T03:25:30.161425-05:DOI: 10.1002/btpr.2601

Authors:Nina Ihling; Natalie Bittner, Sylvia Diederichs, Maximilian Schelden, Anna Korona, Georg Theo Höfler, Alexander Fulton, Karl-Erich Jaeger, Kohsuke Honda, Hisao Ohtake, Jochen BüchsAbstract: Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7-RNA polymerase-dependent Escherichia coli expression strains was compared under inducing and non-inducing conditions. As model enzymes a lipase A from Bacillus subtilis (BSLA) and a 3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus (HBD) were chosen.Four strains were compared during expression of both enzymes in auto-induction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to non-induced second growth phase, thereby ensuring a fair comparison of different strains.In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. This article is protected by copyright. All rights reserved.PubDate: 2018-01-05T03:25:28.050714-05:DOI: 10.1002/btpr.2600

Authors:Zhongming Liu; Dingding Xu, Fangong Kong, Shoujuan Wang, Guihuia Yang, Pedram FatehiAbstract: The main purpose of this paper is to explore the sulfation of xylan to produce an anionic flocculant, sulfated xylan, for removing ethyl violet dye from simulated dye solutions. In this work, xylan was sulfated with chlorosulfonic acid in N, N-dimethylformamide (DMF) solvent and the reaction conditions were optimized using a response surface methodology (RSM). It was observed that the maximum degree of substitution (DS) of 1.1 was obtained for sulfated xylan under the conditions of 3.71 chlorosulfonic acid/xylan molar ratio, 70°C and 7 h reaction time. The resulting sulfated xylan had a charge density of -3.12 mmol/g and molecular weight (Mw) of 22,300 g/mol. Furthermore, elemental and thermogravimetric analyses, Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H-NMR) confirmed the sulfation of xylan. The application of sulfated xylan as a flocculant for decolorizing the simulated ethyl violet dye wastewater was studied. The results indicated that 97% of dye was removed from 50 mg/L dye solution at the sulfated concentration of 175 mg/L and pH 9, but unmodified xylan was ineffective in flocculating and removing dye segments. This article is protected by copyright. All rights reserved.PubDate: 2018-01-02T03:25:22.171467-05:DOI: 10.1002/btpr.2597

Authors:Darryl Sampey; Pascal Courville, David Acree, Jeffrey Hausfeld, William E. BentleyAbstract: The precise product quality and lower cost of goods demands of the growing biosimilars industry are driving biomanufacturing innovation. Biosimilar cell lines that produce complex glycoproteins such as monoclonal antibodies must be both highly productive and express a product with critical quality attributes closely matching those of the innovator reference. In this work, a biomanufacturing platform is described that harnesses the commercially-established NS0 host cell in new ways to create stable, highly productive cell lines with characteristics meeting the current demands. A cholesterol metabolic selection marker and implementation strategy that can be generically applied are shown to yield high expressing cell lines as well as eliminate the need for cholesterol addition, which has been a significant barrier in both stainless steel reactors as well as in single-use plastic systems. Additionally, for the first time, a multiplex selection strategy was implemented that served to increase NS0 cell line specific productivity>10-fold and volumetric yields>6-fold. The best overall performing cell line had a Qp of 28.5 picograms per cell per day was rapidly adapted to a lean production medium. Yields in L-glutamine fed-batch shaker cultures exceeded 500 mg/L. An initial screening of four feeding strategies resulted in a final 13-day yield of over 1.4 g/L in small shaker culture. Overall, this work shows both the strategy to develop biosimilar cell lines and the commercial potential of a novel expression system highly suited for the manufacture of biosimilars of reference biologics currently produced in murine cells. This article is protected by copyright. All rights reserved.PubDate: 2018-01-02T03:20:24.234835-05:DOI: 10.1002/btpr.2596

Authors:Retno Wahyu Nurhayati; Yoshihiro Ojima, Takeaki Dohda, Masahiro Kino-okaAbstract: The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single-use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large-scale production and feasibility of meeting clinical-grade standards. The current work evaluated the capacity of a single-use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (kLa) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h−1, respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7-fold with a total cell number of 1.2±0.2 × 109 cells L−1. In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single-use bioreactor. This article is protected by copyright. All rights reserved.PubDate: 2017-12-11T03:00:36.52466-05:0DOI: 10.1002/btpr.2595

Authors:Karthik Veeravalli; Tony Schindler, Emily Dong, Masaki Yamada, Ryan Hamilton, Michael W. LairdAbstract: Microaerobic (oxygen limited) conditions are advantageous for several industrial applications since a majority of the carbon atoms can be directed for synthesis of desired products. Oxygen limited conditions, however, can result in high levels of undesirable by-products such as acetate which subsequently can have an impact on biomass and product yields. The molecular mechanisms involved in acetate accumulation under oxygen limited conditions are not well understood. Our results indicate that a majority of the genetic modifications known to decrease acetate under aerobic conditions results in similar or even higher acetate under oxygen limitation. Deletion of arcA, whose gene product is a global transcriptional regulator, was the only modification among those evaluated that significantly decreased acetate under both transient and prolonged oxygen limitation. Transcriptome results indicate that the arcA deletion results in an increased expression of the operon involving acs and actP (whose gene products are involved in acetate assimilation and uptake respectively) and some genes in the TCA cycle, thereby promoting increased acetate assimilation. These results provide useful cues for strain design for improved manufacturing of biopharmaceuticals under oxygen limited conditions. This article is protected by copyright. All rights reserved.PubDate: 2017-11-30T03:30:40.165783-05:DOI: 10.1002/btpr.2592

Authors:Marwa I. WahbaAbstract: The poor mechanical stability of chitosan has long impeded its industrial utilization as an immobilization carrier. In this study, the mechanical properties of chitosan beads were greatly improved through utilizing the slow rate of the sodium bicarbonate-induced chitosan gelation and combining it with the chemical cross-linking action of glutaraldehyde (GA). The GA-treated sodium bicarbonate-gelled chitosan beads exhibited much better mechanical properties and up to 2.45-fold higher observed activity of the immobilized enzyme (β-D-galactosidase (β-gal)) when compared to the GA-treated sodium tripolyphosphate (TPP)-gelled chitosan beads. The differences between the sodium bicarbonate-gelled and the TPP-gelled chitosan beads were proven visually and also via scanning electron microscopy, elemental analysis, and differential scanning calorimetry. Moreover, the optimum pH, the optimum temperature, the apparent Km, and the apparent Vmax of the β-gals immobilized onto the two aforementioned types of chitosan beads were determined and compared. A reusability study was also performed. This study proved the superiority of the sodium bicarbonate-gelled chitosan beads as they retained 72.22±4.57% of their initial observed activity during the 13th reusability cycle whereas the TPP-gelled beads lost their activity during the first four reusability cycles, owing to their fragmentation. This article is protected by copyright. All rights reserved.PubDate: 2017-11-29T03:26:57.889278-05:DOI: 10.1002/btpr.2587

Authors:Sean Yu; Pranav Joshi, Yi Ju Park, Kyeong-Nam Yu, Moo-Yeal LeeAbstract: Layer-by-layer cell printing is useful in mimicking layered tissue structures inside the human body and has great potential for being a promising tool in the field of tissue engineering, regenerative medicine, and drug discovery. However, imaging human cells cultured in multiple hydrogel layers in 3D-printed tissue constructs is challenging as the cells are not in a single focal plane. Although confocal microscopy could be a potential solution for this issue, it compromises the throughput which is a key factor in rapidly screening drug efficacy and toxicity in pharmaceutical industries. With epifluorescence microscopy, the throughput can be maintained at a cost of blurred cell images from printed tissue constructs. To rapidly acquire in-focus cell images from bioprinted tissues using an epifluorescence microscope, we created two layers of Hep3B human hepatoma cells by printing green and red fluorescently labeled Hep3B cells encapsulated in two alginate layers in a microwell chip. In-focus fluorescent cell images were obtained in high throughput using an automated epifluorescence microscopy coupled with image analysis algorithms, including three deconvolution methods in combination with three kernel estimation methods, generating a total of nine deconvolution paths. As a result, a combination of Inter-Level Intra-Level Deconvolution (ILILD) algorithm and Richardson-Lucy (RL) kernel estimation proved to be highly useful in bringing out-of-focus cell images into focus, thus rapidly yielding more sensitive and accurate fluorescence reading from the cells in different layers. This article is protected by copyright. All rights reserved.PubDate: 2017-11-29T03:26:51.36976-05:0DOI: 10.1002/btpr.2591

Authors:Glen Giese; Ambrose Williams, Maricel Rodriguez, Josefine PerssonAbstract: Production of knob and hole dual light chain bispecific antibodies poses several unique challenges for development of a feasible industrial scale manufacturing process. We developed an efficient process for the assembly and purification of knob and hole dual light chain bispecific antibodies. Two distinct half-antibodies targeting two different antigens were expressed separately in E. coli cells and captured independently using Protein A chromatography. When combined, the knob and hole mutations in the CH3 domains promoted heterodimer formation. The hinge region disulfides were reduced and re-oxidized to form the disulfide bridge between the two complementary half antibodies. Unreacted half antibodies, non-covalently linked homodimers, covalently linked homodimers, and non-covalently linked heterodimers are impurities closely related to the product of interest and are challenging to remove by standard processes. Characterization of the molecular properties of the half antibodies and high-throughput screening predicted column chromatography performance and allowed for rapid development of downstream purification steps for removal of unique product-related and process-related impurities. This article is protected by copyright. All rights reserved.PubDate: 2017-11-29T03:25:23.904763-05:DOI: 10.1002/btpr.2590

Authors:Jamie Sharp; Tim WGM Spitters, Patrick VermetteAbstract: Few studies report whole pancreatic tissue culture, as it is a difficult task using traditional culture methods. Here, a factorial design was used to investigate the singular and combinational effects of flow, dissolved oxygen concentration (D.O.) and pulsation on whole mechanically-disrupted rat pancreata in a perfusion bioreactor. Whole rat pancreata were cultured for 72h under defined bioreactor process conditions. Secreted insulin was measured and histological (haematoxylin and eosin (H&E)) as well as immunofluorescent insulin staining were performed and quantified. The combination of flow and D.O. had the most significant effect on secreted insulin at 5h and 24h. The D.O. had the biggest effect on tissue histological quality, and pulsation had the biggest effect on the number of insulin-positive structures. Based on the factorial design analysis, bioreactor conditions using high flow, low D.O. and pulsation were selected to further study glucose-stimulated insulin secretion. Here, mechanically-disrupted rat pancreata were cultured for 24h under these bioreactor conditions and were then challenged with high glucose concentration for 6h and high glucose + IBMX (an insulin secretagogue) for a further 6h. These cultures secreted insulin in response to high glucose concentration in the first 6h, however stimulated-insulin secretion was markedly weaker in response to high glucose concentration + IBMX thereafter. After this bioreactor culture period, higher tissue metabolic activity was found compared to that of non-bioreacted static controls. More insulin- and glucagon-positive structures, and extensive intact endothelial structures were observed compared to non-bioreacted static cultures. H&E staining revealed more intact tissue compared to static cultures. This article is protected by copyright. All rights reserved.PubDate: 2017-11-29T03:15:24.635253-05:DOI: 10.1002/btpr.2589

Authors:Ya-Qing Li; Yuan-Shun Liu, Xi-Wang Ying, Hong-Bin Zhou, Zhehua Wang, Sheng-Chang Wu, Jian-Ping Yan, Yu-Ting Jing, Yang YangAbstract: Objective: The aim of the study is to evaluate the effects of silencing a disintegrin and metalloproteinase 17 (ADAM17) gene expression by lentivirus-mediated RNA interference (RNAi) in the gefitinib-resistant lung adenocarcinoma cells, and then to explore whether the recombinant lentivirus mediated ADAM17 RNAi reversed the acquired resistance of lung adenocarcinoma to gefitinib in vitro.Methods: The gefitinib-resistant RPC-9 cells were established and the mutations of EGFR were detected by gene sequencing. The ADAM17 shRNA expression vectors were constructed and packaged to recombinant lentivirus. The cell proliferation viability was detected by MTT, and cellular apotosis was analyzed by flow cytometry assay. The expression levels of ADAM17, EGFR and the phosphorylated EGFR were respectively detected by reverse transcription polymerase chain reaction and western blot. TGF-α production in the supernatant was detected by enzyme-linked immunosorbent assay.Results: The gefitinib-resistant RPC-9 cells in which mutated EGFR (exon 20) carried 790T > T/M mutation were established. When the concentrations of gefitinib were less than 10μmol/L, there were no significant changes in the apoptosis and cellular proliferation of RPC-9 with the dose-escalation of gefitinib. The cell proliferation viability of RPC-9 was significantly decreased by lentivirus mediated ADAM17 RNAi (P 0.05). Gefitinib had no significant effects on TGF alpha production in the supernatants (P > 0.05). Gefitinib did not inhibit EGFR expression in gefitinib-sensitive PC-9 and gefitinib-resistant RPC-9 cells (P > 0.05). The phosphorylation of EGFR in gefitinib-sensitive PC-9 cells was significantly inhibited by gefitinib (P 0.05). Lentivirus mediated ADAM17 RNAi significantly inhibited the mRNA and protein expression of ADAM17 in gefitinib-resistant RPC-9 cells (P PubDate: 2017-10-27T01:10:45.959625-05:DOI: 10.1002/btpr.2564